Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the t...
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MDPI AG
2023-09-01
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| author | Viktoria Sergunova Vladimir Inozemtsev Nina Vorobjeva Elena Kozlova Ekaterina Sherstyukova Snezhanna Lyapunova Aleksandr Chernysh |
| author_facet | Viktoria Sergunova Vladimir Inozemtsev Nina Vorobjeva Elena Kozlova Ekaterina Sherstyukova Snezhanna Lyapunova Aleksandr Chernysh |
| author_sort | Viktoria Sergunova |
| collection | DOAJ |
| description | Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques. |
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| spelling | doaj.art-114e0a64c34149aaa5a1cdbedbbf637a2023-11-19T07:58:36ZengMDPI AGCells2073-44092023-09-011217219910.3390/cells12172199Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy StudyViktoria Sergunova0Vladimir Inozemtsev1Nina Vorobjeva2Elena Kozlova3Ekaterina Sherstyukova4Snezhanna Lyapunova5Aleksandr Chernysh6Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaFederal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaDepartment of Immunology, Biology Faculty, Lomonosov Moscow State University, 119234 Moscow, RussiaFederal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaFederal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaFederal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaFederal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Research Institute of General Reanimatology, 107031 Moscow, RussiaConfocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.https://www.mdpi.com/2073-4409/12/17/2199neutrophilsAFMCLSMNETnanostructuresmembrane |
| spellingShingle | Viktoria Sergunova Vladimir Inozemtsev Nina Vorobjeva Elena Kozlova Ekaterina Sherstyukova Snezhanna Lyapunova Aleksandr Chernysh Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study Cells neutrophils AFM CLSM NET nanostructures membrane |
| title | Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study |
| title_full | Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study |
| title_fullStr | Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study |
| title_full_unstemmed | Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study |
| title_short | Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study |
| title_sort | morphology of neutrophils during their activation and netosis atomic force microscopy study |
| topic | neutrophils AFM CLSM NET nanostructures membrane |
| url | https://www.mdpi.com/2073-4409/12/17/2199 |
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